This research proposal concerns the microsomal membrane bound enzymes which are involved in the activation of the bile acids cholate and deoxycholate with coenzyme A and then their subsequent conjugation with glycine or taurine. The enzymes are (for cholate) cholate:CoA ligase (I), glycine:cholytransferase (II) and taurine:cholytransferase (III). This proposal is aimed at a characterization of the cholate and deoxycholate enzymes and their functional dependence on the membrane lipid environment. The first step will be to carry out for the first time a complete kinetic analysis of the three reactions for each bile acid, using specially developed assay techniques. The hypothesis that reactions (II) and (III) are catalyzed by separate enzymes will be investigated using alternate product and alternate substrate inhibition studies. Further, the question of whether any of the enzymes are common to both cholate and deoxycholate conjugation will be investigated. The basis for the dramatic changes in the ratio of glycine to taurine conjugated bile acids accompanying development and certain diseases will be analyzed by a comparative study of the kinetic parameters of enzymes (II) and (III). The role of the enzymes' membrane lipid environment in determining their enzymatic properties will be determined by selectively modifying the lipid phase by different treatments and by assessing the effects of these treatments on the kinetic properties of the enzymes. An investigation will also be made to determine if the membrane assists the conjugation of bile acids by trapping and concentrating substrates for the enzymes. Since the bile acids are detergents, a study will be made into whether or not in vivo concentrations of bile acid have significant detergent effects on microsomal membranes and their activated enzymes. A comparison will be made between conjugated and unconjugated bile acids to determine if the conjugation process reduces the detergent hazard of bile acids.